397
Biochimica et Biophysica Acta, 521 ( 1 9 7 8 ) 3 9 7 - - 4 0 6 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press
BBA 9 9 2 8 9
GENOME MODIFICATION IN TWO LINES OF BABY HAMSTER KIDNEY FIBROBLASTS (BHK-21)
ANDREW
C.B. CATO, R O G E R
L.P. A D A M S and R O Y H. B U R D O N
Department of Biochemistry, University of Glasgow, Glasgow, G12 8 Q Q (U.K.) (Received March 10th, 1978)
Summary Reasons for the different levels of 5-methyl cytosine encountered in the DNA of two baby hamsters kidney fibroblast lines, BHK-21/C13 and BHK-21/ PyY have been investigated. From enzymic studies it does not seem that there are large numbers of potentially methylatable cytosine residues in the C13 line DNA which contains a lower level of 5-methyl cytosine. Rather it is possible that the difference may be due to the reiteration in the PyY strain of certain sequences containing 5-methyl cytosine which simply occur less frequently in the other line.
Introduction Several lines of evidence have suggested a role for eukaryotic DNA methylation in the control of cellular differentiation (a) the 5-methyl cytosine level of rat liver DNA increases after hydrocortisone administration [1] (b) DNA from different tissues of the same animal has a differing content of 5-methyl cytosine [2--4] (c) the 5-methyl cytosine content of DNA made in the first 2--3 division of the sea urchin embryo rises over several subsequent generations [5], although there is evidence to suggest that it is constant after the 32-cell stage [26]. During an investigation of the level of 5-methyl cytosine in various cultured cell lines it was found that two lines of baby hamster kidney fibroblasts (BHK-21) that were routinely grown in this laboratory showed a particularly large difference in the degree to which their respective DNAs are modified [6]. In view of the various tissue differences described above an investigation into the possible reasons for the differences between the two hamster cell lines was carried out. Abbreviation: Buffer M: 10 mM Tris/HCl pH 7.8/1 mM EDTA/I mM dithiothreitol/10% (v~v) glycerol.
398 Materials and Methods
(i) Growth of cells BHK-21/C13 [7] and BHK-21/PyY [8] (a polyoma virus transformed line) were grown as monolayers in 80-oz. rotating bottles. Each bottle was seeded with 3 • 107 cells and the culture grown for 2 days. Growth medium of Eagle's minimal essential medium (Glasgow modification) supplemented with 10% calf serum (Biocult, Glasgow), 10% tryptose phosphate broth (Difco, bacto), penicilin (100 units/ml) and streptomycin (100 pg/ml). At weekly intervals the cells were screened to eliminate mycoplasmal contamination.
(ii) Preparation o f nuclei Cells were harvested after 2 days of growth and nuclei were prepared by first allowing the cells to swell in 10 mM NaC1/10 mM Tris/HC1 pH 7.4/1.5 mM MgC12 for 10 min. These cells were then resuspended in 1% {v/v) Tween 80 in water, and disrupted by 17 strokes in a Potter-type homogenizer at 4°C. The resulting nuclei were collected by centrifugation at 600 X g for 2 min. These were washed 3 times with 10 mM NaC1/10 mM Tris/HC1 pH 7.4/1.5 mM MgC12 and resuspended in buffer M (50 mM Tris/HC1 pH 7.8/1 mM EDTA/1 mM dithiothreitol/10% {v/v) glycerol).
(iii) DNA methylase preparations Nuclei resuspended in buffer M were made 0.4 M with respect to NaC1 stirred gently for 15 min at 4°C and centrifuged for 1 h at 12 000 Xg to yield a preparation henceforth termed the Nuclear 8upernatant fraction. This fraction was dialysed for 4 h against 2 changes of 100 vols. of buffer M. The dialysed nuclear supernatant fraction was made 32.5--62.5% saturated with ammonium sulphate according to the method of Nottmann et al. [9]. The precipitate containing DNA methylase from this step was collected by centrifugation at 650 × g for 30 min. The pellet was dissolved in 10 ml of buffer M and passed through a column of Sepadex G25 (medium) 6 × 2.5 cm and 3-ml aliquots were collected. The enzyme fraction which eluted in the void volume was loaded onto a DEAE-cellulose column (DE-52, 30 × 1.5 cm previously equilibrated by the passage of 10 vols. of buffer M. The column was washed with 1 vol. of this buffer followed by a linear gradient of buffer M containing 0--0.5 M NaC1. 3-ml aliquots were collected and assayed for DNA methylase activity. The DNA methylase was eluted as a single peak at 0.1 M NaC1 concentration. The pooled enzyme fraction from the DEAE-cellulose column was dialysed for 2 h against 2 1 of buffer M. The dialysed enzyme was then loaded on to a 3 ml hydroxyapatite column; previously equilibrated by passing through it 100 ml of buffer M. The column was washed with 5 vols. of this buffer and the enzyme eluted with a gradient of 0--1 M potassium phosphate in buffer M pH 7.8. The DNA methylase was eluted as a single peak at 0.3 M K2HPO4. The pooled enzyme fraction was again dialysed with 2 changes of 100 vols. of buffer M for 4 h. The enzyme was stored in 0.5-ml portions in plastic ampoules at --70°C until ready to be used. A point of importance with regard to this partial pmd.fication procedure is that throughout, the DNA methylase activity from each BHK-cell type behaved identically.
399
(iv) Enzyme assays (a) DNA methylase assay. The DNA methylase assay was carried out as described by Turnbull and Adams [10] at pH 7.8. Unless otherwise stated the substrate used was E. coli DNA (Sigma) and the reaction incubation time was 1 h, at 37 ° C. (b) Determination of $1 nuclease-sensitive DNA. This was done according to the method of Sutton [11] with slight modifications. E. coil DNA was methylated in vitro by the DNA methylase preparation as described by Turnbull and Adams [ 10]. The methylation reaction was stopped by the addition of 2 ml of a solution containing sodium dodecyl sulphate (1%), EDTA (2 raM), 4-amino salicylic acid (sodium salt) (3%), n-butanol (5%), NaC1 (0.5 M) and 50 pg of heat-denatured sonicated calf thymus DNA. Protein was removed by extraction with phenol/m-cresol/8-hydroxyquinoline, 88 : 10 : 0.1 (v/v/w). After centrifugation at 15°C the upper (aqueous layer) was removed and the DNAs (methylated E. coli DNA and carrier calf thymus DNA) precipitated after mixing with 2 vols. of absolute alcohol. The DNAs were then dissolved in a buffer (0.03 M sodium acetate pH 4.5, 3 . 1 0 -s M ZnSO4, 0.01 M NaC1) and 100 units/ml S~ nuclease (Calbiochem) were added and incubated at 50°C. 100 pl aliquots of the reaction mixture were taken and added to 1 ml of 5% (w/v) cold trichloroacetic acid and the acid-precipitable fraction collected on Whatmann 3 MM filter papers. The filters were washed 3 times with 5% ice-cold trichloroacetic acid and followed by a wash with ethanol and dried in ether. The precipitated DNA on the filters was solubilized by addition of 0.5 ml hyamine hydroxide (1 M solution in methanol) and incubation for 20 min at 60°C. The radioactivity was estimated in toluene scintillator (0.5% diphenyloxazole in toluene. (v) Pyrimidine cluster analysis of DNA methylated enzymatically in vitro by BHK-21/C13 and BHK-21/Py Y cell DNA methylases (a) Depurination and fractionation of oligonucleotides E. coli DNA methylated in vitro by either BHK-21/C13 or BHK-21/PyY cell DNA methylase was mixed with 32P-labelled HeLa cell DNA and depurinated [12]. The resulting oligonucleotides were fractionated in two dimensions as described in ref. 14. 32P-labelled HeLa cell DNA was obtained from HeLa cells labelled with 32PO4 by Drs. K. Vass and B.E.H. Maden of this Department. 32P-labelled HeLa cell DNA was prepared according to the technique of Hell et al [13]. The 32P-labelled HeLa DNA was used in order to facilitate the identification of the oligonucleotides by autoradiography (see ref. 15). (b) Autoradiography. The DEAE-cellulose thin layer plates were placed over Kodirex X-ray films and after storage in lead-lined folders were developed after a week. (c) Estimation of radioactivity. Oligonucleotide-containing areas were excised from the DEAE-cellulose thin layer plates after reference to autoradiographs, (see ref. 6), placed in scintillation vials, treated with hyamine hydroxide as described in Materials and Methods section iv (b) and assessed for their content of 3H and 32p by scintillation spectrometry. (d) Calculation of percentage frequency of 5-methyl cytosine in each oligonucleotide. The frequency of occurrence of 5-methyl cytosine in each oligonucleotide has been expressed relative to the amount of 5-methyl cytosine in
400 the mono-pyrimidine (pCp) fraction as 100, following the procedure outlined by Browne and Burdon [6]. (vi) DNA reassociation kinetics. Initially the BHK-21 cells were labelled either with [6-3H]thymidine (20--30 Ci/mmol) or L-[Me-~4C]methionine (55 Ci/mol) (The Radiochemical Centre, Amersham, U.K.). 5 • 106 cells in 50 ml medium were inoculated into Roux bottles and after 24 h of growth at 37°C either (a) 50 pCi [3H]thymidine added directly or (b) the normal medium was removed and replaced with 50 ml medium containing 50 pCi L-[Me-14C]methionine, 10 mM sodium formate and 2% (v/v) calf serum. After growth for a further 48 h the DNA was extracted by the method of Hell et al. [13] and then sheared by sonication [16] to approximately 200 base pairs. Following the procedure of Young et al. [17], appropriate amounts of the 3H- and 14C-methyl labelled DNAs from each cell type were mixed together, desalted, lyophilised and dissolved in 0.5 M NaC1/25 mM Hepes/0.5 mM EDTA, 50% {v/v) formamide pH 6.8. 50-pl portions were sealed in glass capillaries, heated to 65°C and then incubated at 43°C for different times. The level of reassociation was then assessed on the basis of S1 nuclease digestion again following the procedure of Young et al. [7]. Cot values were computed on the basis that the reassociation of DNA at a concentration of 83 pg • ml -~ for 1 h. corresponds to a Cot value of 1 [18], Results and Discussion
Enzymic properties of BHK cell DNA methylases In a previous report [6] it was shown that in the DNA of BHK-21/C13 cells 1.0% of the cytosines were present as 5-methyl cytosine whereas the corresponding figure for BHK-21/PyY cells was 1.7% Such a difference might simply be due to the activity or sequence specificity of the DNA-methylating enzymes (DNA methylases) in the two cell lines. Since DNA methylases have now been purified from the nuclei of rat liver [19], HeLa cells [20], Krebs II mouse ascites cells [10], and Novikoff hepatoma [21] a partial purification of the methylase activities from both lines of BHK-cells was carried out. Since DNA methylase activity in cultured eukaryotic cells varies with the growth of the cells (maximum DNA methylate activity being found in mid-log phase whilst late-log or early stationary phase cells have less enzyme activity) BHK-21 cells were harvested after mid-log phase. The division time of BHK-21/ C13 and BHK-21/PyY cells is similar although the latter cell type is less contact inhibited [8]. DNA methylase activity was found excusively in nuclei of both cell lines and both methylase activities methylate only cytosine residues in substrate DNA of yield 5-methyl cytosine. The procedure detailed in Materials and Methods resulted in a 200-fold purification of the C13 cell DNA methylase and a 180-fold purification of the PyY cell activity. However the specific activity of the PyY cell preparation was on average at least twice that of the C13 enzyme preparation. Like the DNA methylases from HeLa cells [20], Krebs II cells [10], and Novikoff hepatoma cells [21], the enzymes from both lines of BHK-21 cells were able to methylate "heat-denatured" DNA much better than apparently "native" DNA (Table I).
401 TABLE I METHYL ACCEPTANCE ACTIVITY OF VARIOUS NUCLEIC ACID SUBSTRATES METHYLASE PREPARATIONS FROM BHK-21/C13 CELLS AND BHK-21/PyY CELLS
USING DNA
1 4 0 /J1 o f r e a c t i o n m i x t u r e c o n t a i n e d 4 0 / ~ g D N A , 3 . 3 I~Ci$-adenosyl-L-Me-3H]methionine (1 ~ C i / n m o l ) a n d 2 5 1 ~ g o f B H K - 2 1 / C 1 3 cell D N A m e t h y l a s e o r 4 5 ~g o f B H K - 2 1 / P y Y cell D N A m e t h y l a s e . F o r d e t a i l s o f a s s a y see M a t e r i a l s a n d M e t h o d s . H e a t d e n a t u r a t i o n o f t h e D N A s w a s d o n e a t 1 0 0 ° C f o r 1 0 r a i n in b u f f e r M ( 5 0 m M Tris • HCI p H 7 . 8 / 1 m M E D T A / 1 m M d i t h i o t h r e i t o l / l O % v/v g l y c e r o l ) f o l l o w e d b y rapid cooling. DNA source
Condition
BHK-21/C13 enzyme (dpm/assay per 60 m/n)
BHK-21/PyY enzyme ( d p m / a s s a y p e r 6 0 mill)
E. coli
"Native" Heat-denatured "Native" Heat-denatured "Native" Heat-denatured
1 13 7 12
2 6 7 31
Micrococcus lysodeikticus Calf thymus
776.0 856.0 890.0 282.0 816.4 7 980.0
599.6 139.8 683.6 210.4 677.8 4 634.0
This is somewhat misleading as a close examination of the situation revealed that the methyl groups transferred to the "native"E. coli D N A in fact appeared mainly in regions susceptible to S1-nuclease (i.e.in single-stranded rather than double-stranded regions) (Fig. 1). O n the other hand the small amount of transfer by the P y Y cell enzyme into Si-resistantD N A (Fig. 1) m a y be of significance as the P y Y cell enzyme will methylate both k-phage D N A and ~b X174 D N A whereas the C13 enzyme will only methylate the single-strandedD N A from @ X174 phage. It seems unlikely from these data that the only recognition site for D N A
100
80 '! 60
:~ 20
I
I
I
I
I
I
2
4
6
B
10
12
Time (rain) Fig. 1. S 1 n u c l e a s e d i g e s t i o n o f E. coil D N A m e t h y l a t e d i n v i t r o . 4 0 / ~ g [ 3 H ] m e t h y l - l a b e U e d ( 2 5 0 - - - 3 0 0 d p m / ~ g ) E. coil D N A m e t h y l a t e d i n v i t r o w e r e d i s s o l v e d i n 2 m l o f b u f f e r ( 0 . 0 3 M s o d i u m a c e t a t e , p H 4 . 5 / 3 • 1 0 -S M Z n S O 4 / 0 . 0 1 M NaC1), t o w h i c h w e r e a d d e d 5 0 ~ g o f h e a t - d e n a t u r e d s o n i e a t e d c a l f t h y m u s DNA and 100 unit/m] of S 1 nuclease. This mixture was incubated for various times at 50°C. The reaction w a s s t o p p e d as d e s c r i b e d i n M a t e r i a l s a n d M e t h o d s . • @, S 1 n u c l e a s e d i g e s t i o n o f E. coil D N A m e t h y l a t e d in v i t r o b y D N A m e t h y l a s e f r o m B H K - 2 1 / C 1 3 cells. • . . . . . . 4, S l n u c l e a s e d i g e s t i o n o f E. eoli D N A m e t h y l a t e d in v i t r o b y D N A m e t h y l a s e f r o m B H K - 2 1 / P F Y cells i n t h e p r e s e n c e o f 0 . 1 M N a C I . o o, S 1 n u e l e a s e d i g e s t i o n o f E. coll D N A m e t h y l a t e d i n v i t r o b y D N A m e t h y l a s e f r o m B H K - 2 1 / P y Y cells.
402 .
Biochimica et Biophysica Acta, 521 ( 1 9 7 8 ) 3 9 7 - - 4 0 6 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press
BBA 9 9 2 8 9
GENOME MODIFICATION IN TWO LINES OF BABY HAMSTER KIDNEY FIBROBLASTS (BHK-21)
ANDREW
C.B. CATO, R O G E R
L.P. A D A M S and R O Y H. B U R D O N
Department of Biochemistry, University of Glasgow, Glasgow, G12 8 Q Q (U.K.) (Received March 10th, 1978)
Summary Reasons for the different levels of 5-methyl cytosine encountered in the DNA of two baby hamsters kidney fibroblast lines, BHK-21/C13 and BHK-21/ PyY have been investigated. From enzymic studies it does not seem that there are large numbers of potentially methylatable cytosine residues in the C13 line DNA which contains a lower level of 5-methyl cytosine. Rather it is possible that the difference may be due to the reiteration in the PyY strain of certain sequences containing 5-methyl cytosine which simply occur less frequently in the other line.
Introduction Several lines of evidence have suggested a role for eukaryotic DNA methylation in the control of cellular differentiation (a) the 5-methyl cytosine level of rat liver DNA increases after hydrocortisone administration [1] (b) DNA from different tissues of the same animal has a differing content of 5-methyl cytosine [2--4] (c) the 5-methyl cytosine content of DNA made in the first 2--3 division of the sea urchin embryo rises over several subsequent generations [5], although there is evidence to suggest that it is constant after the 32-cell stage [26]. During an investigation of the level of 5-methyl cytosine in various cultured cell lines it was found that two lines of baby hamster kidney fibroblasts (BHK-21) that were routinely grown in this laboratory showed a particularly large difference in the degree to which their respective DNAs are modified [6]. In view of the various tissue differences described above an investigation into the possible reasons for the differences between the two hamster cell lines was carried out. Abbreviation: Buffer M: 10 mM Tris/HCl pH 7.8/1 mM EDTA/I mM dithiothreitol/10% (v~v) glycerol.
398 Materials and Methods
(i) Growth of cells BHK-21/C13 [7] and BHK-21/PyY [8] (a polyoma virus transformed line) were grown as monolayers in 80-oz. rotating bottles. Each bottle was seeded with 3 • 107 cells and the culture grown for 2 days. Growth medium of Eagle's minimal essential medium (Glasgow modification) supplemented with 10% calf serum (Biocult, Glasgow), 10% tryptose phosphate broth (Difco, bacto), penicilin (100 units/ml) and streptomycin (100 pg/ml). At weekly intervals the cells were screened to eliminate mycoplasmal contamination.
(ii) Preparation o f nuclei Cells were harvested after 2 days of growth and nuclei were prepared by first allowing the cells to swell in 10 mM NaC1/10 mM Tris/HC1 pH 7.4/1.5 mM MgC12 for 10 min. These cells were then resuspended in 1% {v/v) Tween 80 in water, and disrupted by 17 strokes in a Potter-type homogenizer at 4°C. The resulting nuclei were collected by centrifugation at 600 X g for 2 min. These were washed 3 times with 10 mM NaC1/10 mM Tris/HC1 pH 7.4/1.5 mM MgC12 and resuspended in buffer M (50 mM Tris/HC1 pH 7.8/1 mM EDTA/1 mM dithiothreitol/10% {v/v) glycerol).
(iii) DNA methylase preparations Nuclei resuspended in buffer M were made 0.4 M with respect to NaC1 stirred gently for 15 min at 4°C and centrifuged for 1 h at 12 000 Xg to yield a preparation henceforth termed the Nuclear 8upernatant fraction. This fraction was dialysed for 4 h against 2 changes of 100 vols. of buffer M. The dialysed nuclear supernatant fraction was made 32.5--62.5% saturated with ammonium sulphate according to the method of Nottmann et al. [9]. The precipitate containing DNA methylase from this step was collected by centrifugation at 650 × g for 30 min. The pellet was dissolved in 10 ml of buffer M and passed through a column of Sepadex G25 (medium) 6 × 2.5 cm and 3-ml aliquots were collected. The enzyme fraction which eluted in the void volume was loaded onto a DEAE-cellulose column (DE-52, 30 × 1.5 cm previously equilibrated by the passage of 10 vols. of buffer M. The column was washed with 1 vol. of this buffer followed by a linear gradient of buffer M containing 0--0.5 M NaC1. 3-ml aliquots were collected and assayed for DNA methylase activity. The DNA methylase was eluted as a single peak at 0.1 M NaC1 concentration. The pooled enzyme fraction from the DEAE-cellulose column was dialysed for 2 h against 2 1 of buffer M. The dialysed enzyme was then loaded on to a 3 ml hydroxyapatite column; previously equilibrated by passing through it 100 ml of buffer M. The column was washed with 5 vols. of this buffer and the enzyme eluted with a gradient of 0--1 M potassium phosphate in buffer M pH 7.8. The DNA methylase was eluted as a single peak at 0.3 M K2HPO4. The pooled enzyme fraction was again dialysed with 2 changes of 100 vols. of buffer M for 4 h. The enzyme was stored in 0.5-ml portions in plastic ampoules at --70°C until ready to be used. A point of importance with regard to this partial pmd.fication procedure is that throughout, the DNA methylase activity from each BHK-cell type behaved identically.
399
(iv) Enzyme assays (a) DNA methylase assay. The DNA methylase assay was carried out as described by Turnbull and Adams [10] at pH 7.8. Unless otherwise stated the substrate used was E. coli DNA (Sigma) and the reaction incubation time was 1 h, at 37 ° C. (b) Determination of $1 nuclease-sensitive DNA. This was done according to the method of Sutton [11] with slight modifications. E. coil DNA was methylated in vitro by the DNA methylase preparation as described by Turnbull and Adams [ 10]. The methylation reaction was stopped by the addition of 2 ml of a solution containing sodium dodecyl sulphate (1%), EDTA (2 raM), 4-amino salicylic acid (sodium salt) (3%), n-butanol (5%), NaC1 (0.5 M) and 50 pg of heat-denatured sonicated calf thymus DNA. Protein was removed by extraction with phenol/m-cresol/8-hydroxyquinoline, 88 : 10 : 0.1 (v/v/w). After centrifugation at 15°C the upper (aqueous layer) was removed and the DNAs (methylated E. coli DNA and carrier calf thymus DNA) precipitated after mixing with 2 vols. of absolute alcohol. The DNAs were then dissolved in a buffer (0.03 M sodium acetate pH 4.5, 3 . 1 0 -s M ZnSO4, 0.01 M NaC1) and 100 units/ml S~ nuclease (Calbiochem) were added and incubated at 50°C. 100 pl aliquots of the reaction mixture were taken and added to 1 ml of 5% (w/v) cold trichloroacetic acid and the acid-precipitable fraction collected on Whatmann 3 MM filter papers. The filters were washed 3 times with 5% ice-cold trichloroacetic acid and followed by a wash with ethanol and dried in ether. The precipitated DNA on the filters was solubilized by addition of 0.5 ml hyamine hydroxide (1 M solution in methanol) and incubation for 20 min at 60°C. The radioactivity was estimated in toluene scintillator (0.5% diphenyloxazole in toluene. (v) Pyrimidine cluster analysis of DNA methylated enzymatically in vitro by BHK-21/C13 and BHK-21/Py Y cell DNA methylases (a) Depurination and fractionation of oligonucleotides E. coli DNA methylated in vitro by either BHK-21/C13 or BHK-21/PyY cell DNA methylase was mixed with 32P-labelled HeLa cell DNA and depurinated [12]. The resulting oligonucleotides were fractionated in two dimensions as described in ref. 14. 32P-labelled HeLa cell DNA was obtained from HeLa cells labelled with 32PO4 by Drs. K. Vass and B.E.H. Maden of this Department. 32P-labelled HeLa cell DNA was prepared according to the technique of Hell et al [13]. The 32P-labelled HeLa DNA was used in order to facilitate the identification of the oligonucleotides by autoradiography (see ref. 15). (b) Autoradiography. The DEAE-cellulose thin layer plates were placed over Kodirex X-ray films and after storage in lead-lined folders were developed after a week. (c) Estimation of radioactivity. Oligonucleotide-containing areas were excised from the DEAE-cellulose thin layer plates after reference to autoradiographs, (see ref. 6), placed in scintillation vials, treated with hyamine hydroxide as described in Materials and Methods section iv (b) and assessed for their content of 3H and 32p by scintillation spectrometry. (d) Calculation of percentage frequency of 5-methyl cytosine in each oligonucleotide. The frequency of occurrence of 5-methyl cytosine in each oligonucleotide has been expressed relative to the amount of 5-methyl cytosine in
400 the mono-pyrimidine (pCp) fraction as 100, following the procedure outlined by Browne and Burdon [6]. (vi) DNA reassociation kinetics. Initially the BHK-21 cells were labelled either with [6-3H]thymidine (20--30 Ci/mmol) or L-[Me-~4C]methionine (55 Ci/mol) (The Radiochemical Centre, Amersham, U.K.). 5 • 106 cells in 50 ml medium were inoculated into Roux bottles and after 24 h of growth at 37°C either (a) 50 pCi [3H]thymidine added directly or (b) the normal medium was removed and replaced with 50 ml medium containing 50 pCi L-[Me-14C]methionine, 10 mM sodium formate and 2% (v/v) calf serum. After growth for a further 48 h the DNA was extracted by the method of Hell et al. [13] and then sheared by sonication [16] to approximately 200 base pairs. Following the procedure of Young et al. [17], appropriate amounts of the 3H- and 14C-methyl labelled DNAs from each cell type were mixed together, desalted, lyophilised and dissolved in 0.5 M NaC1/25 mM Hepes/0.5 mM EDTA, 50% {v/v) formamide pH 6.8. 50-pl portions were sealed in glass capillaries, heated to 65°C and then incubated at 43°C for different times. The level of reassociation was then assessed on the basis of S1 nuclease digestion again following the procedure of Young et al. [7]. Cot values were computed on the basis that the reassociation of DNA at a concentration of 83 pg • ml -~ for 1 h. corresponds to a Cot value of 1 [18], Results and Discussion
Enzymic properties of BHK cell DNA methylases In a previous report [6] it was shown that in the DNA of BHK-21/C13 cells 1.0% of the cytosines were present as 5-methyl cytosine whereas the corresponding figure for BHK-21/PyY cells was 1.7% Such a difference might simply be due to the activity or sequence specificity of the DNA-methylating enzymes (DNA methylases) in the two cell lines. Since DNA methylases have now been purified from the nuclei of rat liver [19], HeLa cells [20], Krebs II mouse ascites cells [10], and Novikoff hepatoma [21] a partial purification of the methylase activities from both lines of BHK-cells was carried out. Since DNA methylase activity in cultured eukaryotic cells varies with the growth of the cells (maximum DNA methylate activity being found in mid-log phase whilst late-log or early stationary phase cells have less enzyme activity) BHK-21 cells were harvested after mid-log phase. The division time of BHK-21/ C13 and BHK-21/PyY cells is similar although the latter cell type is less contact inhibited [8]. DNA methylase activity was found excusively in nuclei of both cell lines and both methylase activities methylate only cytosine residues in substrate DNA of yield 5-methyl cytosine. The procedure detailed in Materials and Methods resulted in a 200-fold purification of the C13 cell DNA methylase and a 180-fold purification of the PyY cell activity. However the specific activity of the PyY cell preparation was on average at least twice that of the C13 enzyme preparation. Like the DNA methylases from HeLa cells [20], Krebs II cells [10], and Novikoff hepatoma cells [21], the enzymes from both lines of BHK-21 cells were able to methylate "heat-denatured" DNA much better than apparently "native" DNA (Table I).
401 TABLE I METHYL ACCEPTANCE ACTIVITY OF VARIOUS NUCLEIC ACID SUBSTRATES METHYLASE PREPARATIONS FROM BHK-21/C13 CELLS AND BHK-21/PyY CELLS
USING DNA
1 4 0 /J1 o f r e a c t i o n m i x t u r e c o n t a i n e d 4 0 / ~ g D N A , 3 . 3 I~Ci$-adenosyl-L-Me-3H]methionine (1 ~ C i / n m o l ) a n d 2 5 1 ~ g o f B H K - 2 1 / C 1 3 cell D N A m e t h y l a s e o r 4 5 ~g o f B H K - 2 1 / P y Y cell D N A m e t h y l a s e . F o r d e t a i l s o f a s s a y see M a t e r i a l s a n d M e t h o d s . H e a t d e n a t u r a t i o n o f t h e D N A s w a s d o n e a t 1 0 0 ° C f o r 1 0 r a i n in b u f f e r M ( 5 0 m M Tris • HCI p H 7 . 8 / 1 m M E D T A / 1 m M d i t h i o t h r e i t o l / l O % v/v g l y c e r o l ) f o l l o w e d b y rapid cooling. DNA source
Condition
BHK-21/C13 enzyme (dpm/assay per 60 m/n)
BHK-21/PyY enzyme ( d p m / a s s a y p e r 6 0 mill)
E. coli
"Native" Heat-denatured "Native" Heat-denatured "Native" Heat-denatured
1 13 7 12
2 6 7 31
Micrococcus lysodeikticus Calf thymus
776.0 856.0 890.0 282.0 816.4 7 980.0
599.6 139.8 683.6 210.4 677.8 4 634.0
This is somewhat misleading as a close examination of the situation revealed that the methyl groups transferred to the "native"E. coli D N A in fact appeared mainly in regions susceptible to S1-nuclease (i.e.in single-stranded rather than double-stranded regions) (Fig. 1). O n the other hand the small amount of transfer by the P y Y cell enzyme into Si-resistantD N A (Fig. 1) m a y be of significance as the P y Y cell enzyme will methylate both k-phage D N A and ~b X174 D N A whereas the C13 enzyme will only methylate the single-strandedD N A from @ X174 phage. It seems unlikely from these data that the only recognition site for D N A
100
80 '! 60
:~ 20
I
I
I
I
I
I
2
4
6
B
10
12
Time (rain) Fig. 1. S 1 n u c l e a s e d i g e s t i o n o f E. coil D N A m e t h y l a t e d i n v i t r o . 4 0 / ~ g [ 3 H ] m e t h y l - l a b e U e d ( 2 5 0 - - - 3 0 0 d p m / ~ g ) E. coil D N A m e t h y l a t e d i n v i t r o w e r e d i s s o l v e d i n 2 m l o f b u f f e r ( 0 . 0 3 M s o d i u m a c e t a t e , p H 4 . 5 / 3 • 1 0 -S M Z n S O 4 / 0 . 0 1 M NaC1), t o w h i c h w e r e a d d e d 5 0 ~ g o f h e a t - d e n a t u r e d s o n i e a t e d c a l f t h y m u s DNA and 100 unit/m] of S 1 nuclease. This mixture was incubated for various times at 50°C. The reaction w a s s t o p p e d as d e s c r i b e d i n M a t e r i a l s a n d M e t h o d s . • @, S 1 n u c l e a s e d i g e s t i o n o f E. coil D N A m e t h y l a t e d in v i t r o b y D N A m e t h y l a s e f r o m B H K - 2 1 / C 1 3 cells. • . . . . . . 4, S l n u c l e a s e d i g e s t i o n o f E. eoli D N A m e t h y l a t e d in v i t r o b y D N A m e t h y l a s e f r o m B H K - 2 1 / P F Y cells i n t h e p r e s e n c e o f 0 . 1 M N a C I . o o, S 1 n u e l e a s e d i g e s t i o n o f E. coll D N A m e t h y l a t e d i n v i t r o b y D N A m e t h y l a s e f r o m B H K - 2 1 / P y Y cells.
402 .
